The Epidemiology Branch is conducting a number of birth defect studies in collaboration with the Health Research Board and Trinity College, Dublin, Ireland. The main objective of these studies is to determine the relationship between folate and birth defects. The birth defects studied to date are neural tube defects (NTDs), oral clefts, congenital heart defects, Down syndrome and omphalocele. These studies focus on biochemical factors in the area of folate metabolism, and on genetic mutations in folate related genes associated with birth defects. Individual studies of the genetics of neural tube defects (NTDs) contain results on a small number of genes in each report. To identify genetic risk factors for NTDs, we evaluated potentially functional single nucleotide polymorphisms (SNPs) that are biologically plausible risk factors for NTDs but that have never been investigated for an association with NTDs, examined SNPs that previously showed no association with NTDs in published studies, and tried to confirm previously reported associations in folate-related and non-folate-related genes. We investigated 64 SNPs in 34 genes for association with spina bifida in up to 558 case families (520 cases, 507 mothers, 457 fathers) and 994 controls in Ireland. Case-control and mother-control comparisons of genotype frequencies, tests of transmission disequilibrium, and log-linear regression models were used to calculate effect estimates. Spina bifida was associated with over-transmission of the LEPR (leptin receptor) rs1805134 minor C allele genotype relative risk (GRR): 1.5;95% confidence interval (CI): 1.0-2.1;P = 0.0264 and the COMT (catechol-O-methyltransferase) rs737865 major T allele (GRR: 1.4;95% CI: 1.1-2.0;P = 0.0206). After correcting for multiple comparisons, these individual test P-values exceeded 0.05. Consistent with previous reports, spina bifida was associated with MTHFR 677C>T, T (Brachyury) rs3127334, LEPR K109R, and PDGFRA promoter haplotype combinations. The associations between LEPR SNPs and spina bifida suggest a possible mechanism for the finding that obesity is a NTD risk factor. The association between a variant in COMT and spina bifida implicates methylation and epigenetics in NTDs. Neural tube defects (NTDs) are common birth defects (1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C >T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk. A tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case-control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects. Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p <0.01 level. The ten strongest association signals (p-value range: 0.0003-0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 1.23-2.08, p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing. To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk. Future investigations will explore genetic factors that have been shown to affect micronutrients of interest in our quantitative traits genome wide assocation study. For example, we have measured serum and red cell folate levels in over 2500 student volunteers and have generated genome wide data on their genetic variants. These data sets will be merged to determine the most important genetic factors that influence folate levels controlling for use of folic acid containing supplements and fortified food. These results will inform our investigation of genetic factors that are associated with neural tube defect risk.

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Brosnan, John T; Mills, James L; Ueland, Per M et al. (2018) Lifestyle, metabolite, and genetic determinants of formate concentrations in a cross-sectional study in young, healthy adults. Am J Clin Nutr 107:345-354
Mills, James L; Molloy, Anne M; Reynolds, Edward H (2018) Do the benefits of folic acid fortification outweigh the risk of masking vitamin B12 deficiency? BMJ 360:k724
Molloy, Anne M; Mills, James L (2018) Fortifying food with folic acid to prevent neural tube defects: are we now where we ought to be? Am J Clin Nutr 107:857-858
Zhang, Wei; Liu, Aiyi; Albert, Paul S et al. (2018) A pooling strategy to effectively use genotype data in quantitative traits genome-wide association studies. Stat Med 37:4083-4095
Chiu, Chi-Yang; Jung, Jeesun; Chen, Wei et al. (2017) Meta-analysis of quantitative pleiotropic traits for next-generation sequencing with multivariate functional linear models. Eur J Hum Genet 25:350-359
Zhang, Wei; Yang, Liu; Tang, Larry L et al. (2017) GATE: an efficient procedure in study of pleiotropic genetic associations. BMC Genomics 18:552
Chiu, Chi-Yang; Jung, Jeesun; Wang, Yifan et al. (2017) A comparison study of multivariate fixed models and Gene Association with Multiple Traits (GAMuT) for next-generation sequencing. Genet Epidemiol 41:18-34
Velkova, Aneliya; Diaz, Jennifer E L; Pangilinan, Faith et al. (2017) The FUT2 secretor variant p.Trp154Ter influences serum vitamin B12 concentration via holo-haptocorrin, but not holo-transcobalamin, and is associated with haptocorrin glycosylation. Hum Mol Genet 26:4975-4988
Mills, James L; Dimopoulos, Aggeliki (2016) Authors' reply to Smith and colleagues. BMJ 352:i746
Szymczak, Silke; Holzinger, Emily; Dasgupta, Abhijit et al. (2016) r2VIM: A new variable selection method for random forests in genome-wide association studies. BioData Min 9:7

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